Name: isolation, characterization, and purification of macrophages from tissues affected by obesity-related inflammation
Uploaded: 2017-04-03T14:00:00
Description: Watch this Scientific Journal Video about Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation at JoVE.com

ペンシルベニア州立大学ミレニアム科学複雑な流れの Cytometry 中核施設に感謝したいと思います。

すべての実験的プロトコル (セクション 1、1.2、および 1.3) 承認機関動物ケアおよび使用委員会 (IACUC) ペンシルベニア州立大学で。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>組織</td>
			<td>解離バッファー準備</td>
			<td>最終巻</td>
			<td>ストレージ</td>
		</tr>
<tr>
<td>白色脂肪組織 (WAT)</td>
			<td>
解離バッファー: 2.5 %hepes、10 mg/mL ウ...

<ol>
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病気の進行の分子メカニズムを理解するアテローム性動脈硬化、単純な脂肪肝、肝炎などの併存を模倣し、タイプ 2 の糖尿病の食事誘発代謝性疾患モデルが採用されます。コラゲナーゼの消化力依存は細胞外マトリックス (ECM)16,27から細胞を解放するために組織を分離するによく使用されます。コラゲナーゼなどの酵素は、隣接する細胞の構造的な...

マウス モデルは、人間の病気の研究に広く使用されています。病気の状態のマウスから常駐細胞の適切な分離は、病1の病態を分子・細胞の貢献を理解するためのプラットフォームを提供できます。重要です 1 つの障害は、肥満です。肥満の発生率は、インスリン抵抗性と並行世界的に上昇、タイプ 2 の糖尿病、心血管疾患、脂肪肝疾患2,3を継続します。過剰栄養摂取はさらに減らされた身体活動4大動脈および肝臓などの末梢組織の細胞環境を変更することができます脂肪組織から発せられる変更された信号をトリガーによって偏っています。代謝恒常性のような混乱は、慢性低学年全身性炎症5の結果します。
古典的活性化マクロファージの白色脂肪組織 (WAT) の募集と同様に大動脈および肝臓に居住者だけでなく代謝シグナルの調節不全を開始、また炎症6,7を維持する示されています。大食細胞の表現型および機能の多様性は肥満の病因に強く関連付けられている関連する併存7。マクロファージ偏波の動的塑性進行を調整するアクティブ化された表現型の範囲と炎症8の解像度を展示するこれらの細胞が可能です。一方、古典的活性化 (M1) マクロファージは炎症の進展に関与している、あるいはマクロファージ (M2) が解像度と組織修復9,10に関連付けられています。
体は代謝ストレスを受けると白色脂肪組織が異常蓄積されます。拡張の脂肪組織を引き付ける、インスリン抵抗性、高血糖、最終的に 2 型糖尿病、インスリン抵抗性や高血糖11を促進するために通常の脂肪細胞機能を深く変更炎症細胞を保持 12。並行して、白色脂肪組織改造によって解放の炎症性反応では古典的活性化 (M1) 脂肪組織マクロファージ (Atm)13,14を浸透させた。この多細胞器官は肝4大動脈などの他の臓器の正常な機能を derails シグナルのカスケードを発揮します。
肝臓は、近くの調節不全ワット15発刺激への応答の適応代謝パワーハウスです。肝マクロファージまたは代謝の変化に対応した、クッパー細胞実質の両方を変換する炎症性サイトカインと非実質細胞表現型を分泌して組織リモデリングを促進します。肝臓への脂肪蓄積、炎症、過剰な細胞外マトリックス預金、壊死、肝臓障害の広い範囲に貢献して炎症性の侮辱は、非アルコール性脂肪性肝疾患に関連付けられている最終的な関数の損失次のよう16,17,18。
並行して侵害されたワットと肝機能、太い動脈累積動脈壁内の脂質、体は慢性的な代謝ストレス19を受けます。動脈の脂質の蓄積は、活性化された血管内皮細胞によるケモカイン分泌および単球20の後の採用をトリガーします。募集、一度単球増殖を区別するため、リポ蛋白を摂取、泡沫細胞になります。粥状動脈硬化を開始され、プロ炎症性活動による持続的な募集と組織マクロファージ脂質荷を積んだ。この動脈硬化の微小環境の中継細胞外と細胞内ストレス信号に屈して、これらマクロファージは、アポトーシスのシグナル伝達カスケードに従事しています。これらの泡の細胞が死ぬと、プラーク破裂、心筋梗塞や脳卒中につながる病巣の壊死性コアに満たされた脂質内容を貢献します。
総称して、マクロファージの表現型の多様性は一部ワット、肝臓、大動脈8,21などの調節不全組織における炎症性変化による肥満を調整します。特性の採用し、組織マクロファージは、大食細胞の表現型の1を操作する分子標的の可能性への洞察を提供すること。効果的に炎症を起こした組織の肥満によるマクロファージを特徴付ける、酵素消化による単一細胞懸濁液が得られます。このような分離のプロトコルは免疫細胞死を最小限に抑えながら、最適な細胞収率を提供する結合組織を十分に分解に効果的である必要があります。酵素の混合、組織とその構造のメイクアップの種類に依存します。大動脈など弾力性のある組織では、肝組織の解離を達成するためにワットと比較してより強力な酵素活性を必要があります。単一細胞懸濁液から組織マクロファージが明確に特徴するまたは、転写プロファイリングなどさらに下流解析の分離します。
ここで組織固有のプロトコルは説明を効果的に分離し、伝統的な食生活による肥満、住民組織マクロファージを特徴付けるコラゲナーゼ依存組織消化と多色フローサイトメトリーを使用します。アテローム性動脈硬化、単純な脂肪肝と肝炎マウス モデル。同時白血球 - (CD45 や CD11b) やマクロファージに対する抗体と細胞表面のマーカーの染色 (F4/80) 特定の抗原は、しばしばマクロファージ人口22を識別するために使用されます。蛍光活性化セル (FACS) を並べ替えは、高純度でこれらの特定の集団を並べ替えるために使用する強力な戦略です。並べ替えられた人口は、表現型特異的遺伝子プロファイル (量的なリアルタイムのポリメラーゼの連鎖反応) などの下流の分子分析23を使用して評価できます。標準フローサイトメトリー ・ フロー フローサイトメトリーを用いた細胞のソーティングは非常に異種細胞懸濁液内マクロファージを区別する強力なツールであるが、正常な出力を確保するため元のプロトコルを最適化する必要があります。本研究では効果的に分離し、実行可能なティッシュ特定の大食細胞を特徴付けるプロトコルを説明します。もっと重要なは、この研究は、しばしば発生を防ぐおよび/またはそれらを解決するプロアクティブとトラブルシューティング戦略だけでなく、技術的な問題に重要な洞察を提供します。

<table border="0" cellpadding="0" cellspacing="0" width="940">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">26G x 5/8 in Needles</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">305115</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">23G x 0.75 in needle x 12 in. tubing Blood Collection Set</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">367297</td>
			<td style="width:349px;">Used for cannulation of subhepatic IVC during liver perfusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">21G 1 1/2 in. Needles</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">305156</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">1mL syringe with rubber stops</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">309659</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">10mL Syringes</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">309604</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">1mL Syringe</td>
			<td style="width:151px;">BD</td>
			<td style="width:119px;">309659</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">F4/80 PE</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">123110</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">CD11c PE/Cy7</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">117318</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">CD11b PE/Cy5</td>
			<td style="width:151px;">eBioscience</td>
			<td style="width:119px;">15-0112-81</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Anti-mouse CD16/32 Fc Block&#160;</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">101320</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">CD45 Pacific Blue</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">103126</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">PE Rat IgG2a</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">400508</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">PE/Cy7 Armenian Hamster IgG</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">400922</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">PE/Cy5 Rat IgG2b</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">400610</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Pacific Blue Rat IgG2c</td>
			<td style="width:151px;">Biolegend</td>
			<td style="width:119px;">400717</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Dulbecco&#8217;s Modified Eagle Medium (DMEM)&#160;</td>
			<td style="width:151px;">Cellgro</td>
			<td style="width:119px;">15-017-CV</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">1X Phosphate Buffered Saline (PBS)</td>
			<td style="width:151px;">Cellgro</td>
			<td style="width:119px;">21-031-CV</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">70 micron cell strainers</td>
			<td style="width:151px;">Corning, Inc.</td>
			<td style="width:119px;">352350</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">&#160;1.7 mL microcentrifuge tube&#160;</td>
			<td style="width:151px;">Denville</td>
			<td style="width:119px;">C2170</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">Paraformaldehyde Aqueous Solution -16X</td>
			<td style="width:151px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">CAS #30525-89-4</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">Micro Dissecting Scissors, 3.5 inch, Straight, 23mm, Sharp&#160;</td>
			<td>Stoelting</td>
			<td>52132-10P</td>
			<td style="width:349px;">Used for general dissecting purposes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Forceps, 4in, full curve, 0.8mm</td>
			<td>Stoelting</td>
			<td>52102-37P&#160;</td>
			<td style="width:349px;">Used for general dissecting purposes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Spring dissection scissors &#8211; 3 mm Cutting edge&#160;</td>
			<td style="width:151px;">Fine Science Tool</td>
			<td style="width:119px;">15000-10</td>
			<td style="width:349px;">Used for aorta dissection Steps 1.3.3.17 to 1.3.3.28</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Curved 0.07 x 0.04 mm Tip Forceps&#160;</td>
			<td style="width:151px;">Fine Science Tool</td>
			<td style="width:119px;">11297-10</td>
			<td style="width:349px;">Used for aorta dissection Steps 1.3.3.17 to 1.3.3.28</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Hemostatic Forceps (Curved)</td>
			<td style="width:151px;">Fine Science Tool</td>
			<td style="width:119px;">13021-12</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Heparin Sodium Salt</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">9041-08-1</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">35mm Cell Culture/Petri Dishes</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">12-565-90</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Polystyrene Petri Dishes (10 cm) w/lid</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">08-757-100D</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">15mL Conical Centrifuge Tubes (Polypropylene)</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">14-959-53A</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">50mL Conical Centrifuge Tubes&#160;(Polypropylene)</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">14-432-22</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">5mL Round-Bottom Polystyrene Tubes&#160;</td>
			<td style="width:151px;">Fischer Scientific</td>
			<td style="width:119px;">14-959-5</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Fetal Bovine Serum</td>
			<td style="width:151px;">Gemini Bio-Products</td>
			<td style="width:119px;">100-106</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:321px;">Ethanol (Stock Ethyl Alcohol Denatured, Anyhydrous)</td>
			<td style="width:151px;">Millipore</td>
			<td style="width:119px;">EX0285-1</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Bovine Serum Albumin</td>
			<td style="width:151px;">Rockland</td>
			<td style="width:119px;">BSA-50</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">HEPES</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3375</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Collagenase Type II</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">C6885</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Collagenasse Type XI</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">C7657</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Hyaluronidase Type I</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">H3506</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">DNAse</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">DN25</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Collagenase Type I</td>
			<td style="width:151px;">Sigma-Aldrich</td>
			<td style="width:119px;">C0130</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">NaOH</td>
			<td style="width:151px;">Sigma Aldrich</td>
			<td style="width:119px;">1310-73-2&#160;</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:321px;">CaCl2</td>
			<td style="width:151px;">Sigma Aldrich</td>
			<td style="width:119px;">449709-10G</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">500mL beaker&#160;</td>
			<td style="width:151px;">Sigma Aldrich</td>
			<td style="width:119px;">02-540M</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">4 cm Hemostatic clamp</td>
			<td style="width:151px;">Stoelting</td>
			<td style="width:119px;">52120-40</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Toothed forceps</td>
			<td style="width:151px;">Stoelting</td>
			<td style="width:119px;">52104-33P</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">50 micron Disposable filters</td>
			<td style="width:151px;">Systemex</td>
			<td style="width:119px;">04-0042-2317</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">Collagenase Type IV</td>
			<td style="width:151px;">ThermoFischer Scientific</td>
			<td style="width:119px;">17104019</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">Ammonium Chloride Potassium (ACK)</td>
			<td style="width:151px;">ThermoFischer Scientific&#160;</td>
			<td style="width:119px;">A1049201</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Razors (0.22 mm (0.009&#34;))</td>
			<td style="width:151px;">VWR International</td>
			<td style="width:119px;">55411-050</td>
			<td style="width:349px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Texas Red Live/Dead stain</td>
			<td style="width:151px;"></td>
			<td style="width:119px;"></td>
			<td style="width:349px;">Red viability stain (in Figure 1A)</td>
		</tr>
	</tbody>
</table>

isolation, characterization, and purification of macrophages from tissues affected by obesity-related inflammation

肥満は、主として単球由来マクロファージと同様に、住民組織マクロファージによる慢性的な炎症状態を促進します。食餌誘導性肥満 (DIO) はマクロファージの多様性; の役割を勉強の貴重なモデルただし、分離十分なマクロファージは炎症を起こした組織から取得する困難です。このプロトコルでは、私達は分離手順と必要なトラブルシューティングのガイドライン 18 週間の高脂肪 (HFD) または高脂肪高コレステロール (マウスから住民組織マクロファージの適切な人口を得るため私たちの研究から派生した輪郭を描くHFHCD) 食事介入。このプロトコルは、肥満や動脈硬化、肝、白色脂肪組織 (WAT)、大動脈などで学んだ 3 つのホールマーク組織に焦点を当てください。我々 はどのように二元的な使用を強調流れの cytometry 分離の新しいディメンションと住民組織マクロファージの性質を達成できます。このプロトコルの基本的なセクションは、フローサイトメトリーによる解析の染色後の細胞表面抗体とマクロファージの分離、組織固有の酵素の消化力の基になる複雑さを扱います。このプロトコルは、蛍光活性化セル (FACS) を並べ替えの基になる既存の複雑さに対処し、適切に並べ替えられた細胞集団から幅広い評価を得られるようにこれらの複雑さへの明確化を提案します。豊かな代替メソッドは、並べ替え FACS を操作するときの柔軟性と時間の管理を可能にする高密度の肝臓などの細胞に含まれています。簡単に言えば、このプロトコルを特定の研究では炎症を起こした組織の多数のマクロファージの不均一性を評価する研究者を助けるし、良好な細胞の単離と解析の成功している洞察力のトラブルシューティングのヒントを提供しますDIO を介した炎症における免疫細胞。

アポリポ蛋白 E 欠損 (アポ島) C57BL/6 (BL6) マウス 18 週間、1 x 10 の4 - 2 × 104 CD45 (HCHFD または HCD) 高脂肪高コレステロール食で維持を使用する場合+F4/80+ 2 つのサンプルは、大動脈マクロファージが分離することができますプール。HFHCD 給電アポ KO マウスの胃から肝臓生産 (これは、使用可能な並べ替え時間異なります) 5 x 105並べ?...

Watch this Scientific Journal Video about Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation at JoVE.com

Isolation, Characterization, and Purification of Macrophages from Tissues Affected by Obesity-related Inflammation

このプロトコルにより、研究者を分離し、特徴付ける組織居住者様々 なマクロファージ ホールマーク食事誘発代謝疾患モデルから抽出された組織の炎症。

肥満に関連する炎症によって影響を受ける組織のマクロファージの精製と性質、分離

Obesity promotes a chronic inflammatory state that is largely mediated by tissue-resident macrophages as well as monocyte-derived macrophages. ...

The overall goal of this procedure is to effectively isolate and characterize macrophages from tissues commonly affected by diet-induced inflammation such as the liver. This method can help answer key questions in the fields of immunology and inflammation about how tissue-resident macrophage phenotype can dictate the progression of chronic inflammatory diseases. The main advantage of this is that the procedure optimizes the isolation steps needed for obtaining single-cell suspensions derived from tissues impacted by obesity-mediated low-grade chronic inflammation.Demonstrating the procedure will be Joselyn Allen, a graduate student from my laboratory. To isolate the liver, first place an ethanol-soaked mouse in the supine position on a polystyrene foam dissecting board and secure the fore and hind paws. Next, use a medium point tip forceps to grasp the abdominal skin near the urethra opening and use sharp dissecting scissors to create a small incision in the raised skin.Insert the lower blade of the scissors into the incision between the skin and the peritoneum and make a lateral incision from the groin to the chin. Gently pull back the skin to expose the intact peritoneal cavity and thoracic wall and make a lateral incision through the peritoneum. Lift the sternum and carefully incise the diaphragm.Then move the gastrointestinal organs to the side and locate the subhepatic inferior vena cava or IVC. Using hemostatic forceps, clamp the suprahepatic IVC to maintain a localized perfusion and attach a syringe filled with pre-warmed perfusion buffer to a 23 gauge blood collection and infusion set. Gently depress the plunger until the tubing and needle are filled with perfusion buffer and insert the needle parallel to the subhepatic IVC.Proper cannulation of the IVC ensures that the dissociation buffer is locally distributed throughout the liver and peripheral tissues optimizing the tissue digestion. Use a four centimeter hemostatic clamp to secure the needle in place and gently depress the plunger to begin the perfusion. If the needle is appropriately positioned, the color will rapidly flush from the liver and the lobes will become enlarged.Promptly cut the portal vein to allow the perfusion buffer to flow freely through the liver, occasionally gently massaging the lobes between the thumb and forefinger to facilitate the removal of the blood. When only five milliliters of perfusion buffer are left, replace the perfusion buffer syringe with a syringe filled with pre-warmed dissociation buffer, continuing to gently massage the lobes and perfuse the liver until it is fully digested. Confirm a successful digestion by gently pressing the blunt edge of a pair of forceps into the left lateral lobe.An indentation should appear on the surface that slowly fills once the forceps is removed. Then remove the needle and use a short straight blade from a pair of dissecting scissors to carefully cut through the connecting ligaments to excise the whole liver and the gallbladder. Transfer the liver to a 10 centimeter dish containing 10 milliliters of ice cold DMEM and use toothed forceps to grip the severed suprahepatic IVC.Using medium point forceps, apply a rapid stroking motion to each lobe to release the cells from the Glisson's Capsule and pass the saturated DMEM through a 70 micrometer cell strainer in a 50 milliliter conical tube on ice. High yields of viable cells can only be achieved by properly dissociating the liver cells from the Glisson's Capsule. When all of the cells have been collected, invert the tube to gently mix the cell suspension and pellet the cells by centrifugation.Transfer the supernatant to a new 50 milliliter conical tube for three more low-spin centrifugations. After the last centrifugation, transfer the supernatant to a new 50 milliliter tube and centrifuge the cells one more time at a higher speed. Then resuspend the non-parenchymal cell pellet in FACS buffer for counting and dilute the liver cells to the appropriate concentration according to their downstream application in labeled five milliliter round bottom polystyrene tubes.Mice fed a high fat diet or a high fat high cholesterol diet exhibit an increased infiltration of classically activated M1 macrophages in affected tissues such as the aorta. RON receptor expressing aortic CD45 positive F4/80 positive macrophages which demonstrate an anti-inflammatory phenotype are decreased in high fat high cholesterol diet fed mice while pro-inflammatory CD45 positive F4/80 positive CD11c positive macrophages are found in increased numbers in aortas isolated from these animals. Further, sorted RON receptor expressing macrophages derived from digested aortas display an increased expression of Arginase 1 gene, a well-established M2 macrophage marker.Indeed, the characterization of liver-resident macrophages reveals the prevailing phenotype of RON receptor expressing subpopulations with CD11c positive pro-inflammatory macrophage populations demonstrating a decreased expression of genes that are strongly associated with the ant-inflammatory M2 phenotype. Similar trends are observed in the macrophage populations isolated from digested white adipose tissue with pro-inflammatory phenotype macrophage populations exhibiting a decreased surface expression of the RON receptor. Once mastered, this technique can be completed in 20 to 30 minutes per animal if it is performed properly.Following this procedure, other methods such as flow cytometry, fluorescent-activated cell sorting, and/or quantitative PCR can be performed to further characterize the phenotype of the isolated liver-derived tissue-resident macrophages. After watching this video, you should have a good understanding of how to properly isolate tissue-resident macrophages from diseased livers. Thanks for watching and good luck with your experiments.

Research

JoVE Journal

Immunology and Infection

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

マウスの腸からの樹状細胞およびマクロファージの単離とキャラクタリゼーション

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

免疫・感染学

Proteomic Profiling of Macrophages by 2D Electrophoresis

2D電気泳動によるマクロファージのプロテオームプロファイリング

The goal of the two-dimensional (2D) electrophoresis protocol described here is to show how to analyse the phenotype of human cultured macrophages. The ...

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

母体·胎児界面におけるマウス組織からの白血球の分離

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen

マウスの脾臓から不変ナチュラルキラーT細胞を単離および拡張するための最適化方法

The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

単離、特徴付けおよび歯肉免疫細胞ネットワークの機能検討

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

Functional Characterization of Regulatory Macrophages That Inhibit Graft-reactive Immunity

グラフト反応性免疫を阻害する調節性マクロファージの機能的特徴付け

Macrophage accumulation in transplanted organs has long been recognized as a feature of allograft rejection1. Immunogenic monocytes infiltrate the ...

Phenotypic Characterization of Macrophages from Rat Kidney by Flow Cytometry

フローサイトメトリーによるラットの腎臓からのマクロファージの表現型の特徴

There is increasing evidence suggesting the important role of inflammation and, subsequently, macrophages in the development and progression of renal ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

フローサイトメトリーによる中枢神経系からのミクログリアおよび単球由来マクロファージの解析

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

サルモネラの分離-大食細胞のファゴゾームを含む

Salmonella typhimurium is a facultative intracellular bacterium that causes gastroenteritis in humans. After invasion of the lamina propria, S. ...

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

Isolation and Characterization of Extracellular Vesicles from Adult Schistosoma japonicum

分離と大人住血吸虫から細胞外小胞のキャラクタリゼーション

Extracellular vesicles (EVs) are membranous vesicles released by a variety of cells into the extracellular microenvironment. EVs represent a population of ...